Field of the Invention
This invention relates to methods and compositions for isolating, detecting, diagnosing and/or characterizing Trop-2+ cancer cells, preferably from the circulation. The methods and compositions utilize anti-Trop-2 antibodies, which may be monovalent, bivalent or multivalent. In a preferred embodiment, anti-Trop-2 antibodies are the sole anti-TAA (tumor-associated antigen) capture antibodies utilized in the assay, which does not include use of mixtures of antibodies against TAAs other than Trop-2. In alternative embodiments, the capture antibody may be a bispecific antibody comprising an anti-Trop-2 antibody or fragment and a second antibody or fragment against a different TAA. More preferably, the antibodies are rodent, chimeric, humanized or human antibodies or antigen-binding fragments thereof. Expression of Trop-2 in cancer cells may be assessed using known techniques, including but not limited to binding of anti-Trop-2 antibodies as detected by flow cytometry or immunohistochemistry, and quantitative RT-PCR. Automated systems and devices that have been developed to isolate and/or detect circulating tumor cells (CTCs), including but not limited to the MagSweeper device (Illumina, Inc., San Diego, Calif.), LIQUIDBIOPSY® system (Cynvenio Biosystems, Inc., Westlake Village, Calif.), CELLSEARCH® system (Vendex LLC, Raritan, N.J.), GILUPI CELLCOLLECTOR™ (GILUPI GmbH, Potsdam, Germany), APOSTREAM® system (Apocell, Houston, Tex.), ONCOCEE™ microfluidic platform (BioCept Laboratories, San Diego, Calif.), VerIFAST System (Casavant et al., 2013, Lab Chip 13:391-6; 2014, Lab Chip 14:99-105) or ISOFLUX™ system (Fluxion, South San Francisco, Calif.) may be utilized in the practice of the claimed methods. Most preferably, the anti-Trop-2 antibody is a murine, chimeric or humanized RS7 (hRS7) antibody, comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:1); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:5) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:6). However, in alternative embodiments other known anti-Trop-2 antibodies may be utilized, as discussed below. The methods and compositions are applicable for the enrichment, isolation, detection, diagnosis and/or characterization of various metastatic Trop-2-expressing cancers, such as breast (e.g., triple-negative breast cancer), ovarian, cervical, endometrial, lung, prostate, colon, rectum, stomach, esophageal, bladder, renal, pancreatic, thyroid, epithelial, and head-and-neck cancers. Anti-Trop-2 antibodies may be utilized in combination with one or more labeled detection antibodies, or may be directly labeled by conjugation with at least one diagnostic agent. Alternatively, a bispecific antibody may comprise one binding site for Trop-2 and another binding site for a hapten on a targetable construct, typically a small peptide labeled with at least one diagnostic agent. In certain alternative embodiments, detection of Trop-2+ CTCs may be followed by therapeutic treatment of the Trop-2+ cancer, using anti-Trop-2 antibodies or fragments thereof. Preferably the antibody or fragment is conjugated to at least one therapeutic agent, such as antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes or radioisotopes. More preferably, the therapeutic agent is SN-38 or P2PDOX.
Related Art
Trop-2 (human trophoblast-cell-surface marker) is a cell surface glycoprotein that was originally identified in normal and malignant trophoblast cells (Lipinski et al., 1981, Proc Natl. Acad Sci USA 78:5147-50). Trop-2 is highly expressed in most human carcinomas, particularly in epithelial carcinomas and adenocarcinomas, with reported low to restricted expression in normal tissues (see, e.g., Cubas et al., 2010, Molec Cancer 9:253; Stepan et al., 2011, J Histochem Cytochem 59:701-10; Varughese et al., 2011, Am J Obst Gyn 205:567e-e7). Expression of Trop-2 is associated with metastasis, increased tumor aggressiveness and decreased patient survival (Cubas et al., 2010; Varughese et al., 2011). Pathogenic effects of Trop-2 have been reported to be mediated, at least in part, by the ERK ½MAPK pathway (Cubas et al., 2010).
It has been proposed that early in tumor progression, cancer cells may be found in low concentration in the circulation (see, e.g., Krishnamurthy et al., 2013, Cancer Medicine 2:226-33; Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Wang et al., Feb. 24, 2015, Int J Clin Oncol, Epub ahead of print). Due to the relatively non-invasive nature of blood sample collection, there has been great interest in the isolation and detection of CTCs, to promote cancer diagnosis at an earlier stage of the disease and as a predictor for tumor progression, disease prognosis and/or responsiveness to drug therapy (see, e.g., Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Winer-Jones et al., 2014, PLoS One 9:e86717; U.S. Patent Appl. Publ. No. 2014/0357659).
Various techniques and apparatus have been developed to isolate and/or detect circulating tumor cells. Several reviews of the field have recently been published (see, e.g., Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Joosse et al., 2014, EMBO Mol Med 7:1-11; Truini et al., 2014, Fron Oncol 4:242). The techniques have involved enrichment and/or isolation of CTCs, generally using capture antibodies against an antigen expressed on tumor cells, and separation with magnetic nanoparticles, microfluidic devices, filtration, magnetic separation, centrifugation, flow cytometry and/or cell sorting devices (e.g., Krishnamurthy et al., 2013, Cancer Medicine 2:226-33; Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Joosse et al., 2014, EMBO Mol Med 7:1-11; Truini et al., 2014, Fron Oncol 4:242; Powell et al., 2012, PLoS ONE 7:e33788; Winer-Jones et al., 2014, PLoS One 9:e86717; Gupta et al., 2012, Biomicrofluidics 6:24133; Saucedo-Zeni et al., 2012, Int J Oncol 41:1241-50; Harb et al., 2013, Transl Oncol 6:528-38). The enriched or isolated CTCs may then be analyzed using a variety of known methods, as discussed further below. Systems or apparatus that have been used for CTC isolation and detection include the CELLSEARCH® system (e.g., Truini et al., 2014, Front Oncol 4:242), MagSweeper device (e.g., Powell et al., 2012, PLoS ONE 7:e33788), LIQUIDBIOPSY® system (Winer-Jones et al., 2014, PLoS One 9:e86717), APOSTREAM® system (e.g., Gupta et al., 2012, Biomicrofluidics 6:24133), GILUPI CELLCOLLECTOR™ (e.g., Saucedo-Zeni et al., 2012, Int J Oncol 41:1241-50), and ISOFLUX™ system (Harb et al., 2013, Transl Oncol 6:528-38).
To date, the only FDA-approved technology for CTC detection involves the CELLSEARCH® platform (Vendex LLC, Raritan, N.J.), which utilizes anti-EpCAM antibodies attached to magnetic nanoparticles to capture CTCs. Detection of bound cells occurs with fluorescent-labeled antibodies against cytokeratin (CK) and CD45. Fluorescently labeled cells bound to magnetic particles are separated out using a strong magnetic field and are counted by digital fluorescence microscopy. The CELLSEARCH® system has received FDA approval for detection of metastatic breast, prostate and colorectal cancers.
Most CTC detection systems have focused on use of anti-EpCAM capture antibodies (see, e.g., Truini et al., 2014, Front Oncol 4:242; Powell et al., 2012, PLoS ONE 7:e33788; Alix-Panabieres & Pantel, 2013, Clin Chem 50:110-18; Lin et al., 2013, Biosens Bioelectron 40:63-67; Wang et al., Feb. 24, 2015, Int J Clin Oncol Epub ahead of print; Magbanua et al., 2015, Clin Cancer Res 21:1098-105; Harb et al., 2013, Transl Oncol 6:528-38). However, not all metastatic tumors express EpCAM (see, e.g., Mikolajcyzyk et al., 2011, J Oncol 2011:252361; Pecot et al., 2011, Cancer Discovery 1:580-86; Gupta et al., 2012, Biomicrofluidics 6:24133). Attempts have been made to utilize alternative schemes for isolating and detecting EpCAM-negative CTCs, such as use of antibody combinations against TAAs. Antibodies against as many as 10 different TAAs have been utilized in an attempt to increase recovery of metastatic circulating tumor cells (e.g., Mikolajcyzyk et al., 2011, J Oncol 2011:252361; Pecot et al., 2011, Cancer Discovery 1:580-86; Krishnamurthy et al., 2013, Cancer Medicine 2:226-33; Winer-Jones et al., 2014, PLoS One 9:e86717).
Drawbacks exist to such approaches, including the complexity of preparing and using large numbers of different antibodies and their attachment to magnetic nanoparticles, microfluidic devices or other separation technologies, as well as potential cross-reactivity against normal cell populations when using a broad spectrum of anti-tumor antibodies. A need exists in the art for improved methods of isolating, detecting, diagnosing and/or characterizing CTCs, using antibodies against a single TAA that is expressed in a broad range of tumors.